Development and characterization of anti-galectin-9 antibodies that protect T cells from galectin-9-induced cell death.

Antibodies that target immune checkpoint proteins such as programmed cell death protein 1, programmed death ligand 1, and cytotoxic T-lymphocyte-associated antigen 4 in human cancers have achieved impressive clinical success; however, a significant proportion of patients fail to respond to these treatments. Galectin-9 (Gal-9), a ß-galactoside-binding protein, has been shown to induce T-cell death and facilitate immunosuppression in the tumor microenvironment by binding to immunomodulatory receptors such as T-cell immunoglobulin and mucin domain-containing molecule 3 and the innate immune receptor dectin-1, suggesting that it may have potential as a target for cancer immunotherapy. Here, we report the development of two novel Gal-9-neutralizing antibodies that specifically react with the N-carbohydrate-recognition domain of human Gal-9 with high affinity. We also show using cell-based functional assays that these antibodies efficiently protected human T cells from Gal-9-induced cell death. Notably, in a T-cell/tumor cell coculture assay of cytotoxicity, these antibodies significantly promoted T cell-mediated killing of tumor cells. Taken together, our findings demonstrate potent inhibition of human Gal-9 by neutralizing antibodies, which may open new avenues for cancer immunotherapy.

Galectin-12 modulates sebocyte proliferation and cell cycle progression by regulating cyclin A1 and CDK2.

Enhanced sebocyte proliferation is associated with the pathogenesis of human skin diseases related to sebaceous gland hyperfunction and androgens, which are known to induce sebocyte proliferation, are key mediators of this process. Galectin-12, a member of the ß-galactoside-binding lectin family that is preferentially expressed by adipocytes and functions as an intrinsic negative regulator of lipolysis, has been shown to be expressed by human sebocytes. In this study, we identified galectin-12 as an important intracellular regulator of sebocyte proliferation. Galectin-12 knockdown in the human SZ95 sebocyte line suppressed cell proliferation, and its overexpression promoted cell cycle progression. Inhibition of galectin-12 expression reduced the androgen-induced SZ95 sebocyte proliferation and growth of sebaceous glands in mice, respectively. The mRNA expression of the key cell cycle regulators cyclin A1 (CCNA1) and cyclin-dependent kinase 2CDK2 was reduced in galectin-12 knockdown SZ95 sebocytes, suggesting a pathway of galectin-12 regulation of sebocyte proliferation. Further, galectin-12 enhanced peroxisome proliferator-activated receptor gamma (PPARγ) expression and transcriptional activity in SZ95 sebocytes, consistent with our previous studies in adipocytes. Rosiglitazone, a PPARγ ligand, induced CCNA1 levels, suggesting that galectin-12 may upregulate CCNA1 expression via PPARγ. Our findings suggest the possibility of targeting galectin-12 to treat human sebaceous gland hyperfunction and androgen-associated skin diseases.

An adipose tissue galectin controls endothelial cell function via preferential recognition of 3-fucosylated glycans.

Upon overnutrition, adipocytes activate a homeostatic program to adjust anabolic pressure. An inflammatory response enables adipose tissue (AT) expansion with concomitant enlargement of its capillary network, and reduces energy storage by increasing insulin resistance. Galectin-12 (Gal-12), an endogenous lectin preferentially expressed in AT, plays a key role in adipocyte differentiation, lipolysis, and glucose homeostasis. Here, we reveal biochemical and biophysical determinants of Gal-12 structure, including its preferential recognition of 3-fucosylated structures, a unique feature among members of the galectin family. Furthermore, we identify a previously unanticipated role for this lectin in the regulation of angiogenesis within AT. Gal-12 showed preferential localization within the inner side of lipid droplets, and its expression was upregulated under hypoxic conditions. Through glycosylation-dependent binding to endothelial cells, Gal-12 promoted in vitro angiogenesis. Moreover, analysis of in vivo AT vasculature showed reduced vascular networks in Gal-12-deficient (Lgals12-/-) compared to wild-type mice, supporting a role for this lectin in AT angiogenesis. In conclusion, this study unveils biochemical, topological, and functional features of a hypoxia-regulated galectin in AT, which modulates endothelial cell function through recognition of 3-fucosylated glycans. Thus, glycosylation-dependent programs may control AT homeostasis by modulating endothelial cell biology with critical implications in metabolic disorders and inflammation.

Galectin-12 in Cellular Differentiation, Apoptosis and Polarization.

Galectin-12 is a member of a family of mammalian lectins characterized by their affinity for ß-galactosides and consensus amino acid sequences. The protein structure consists of a single polypeptide chain containing two carbohydrate-recognition domains joined by a linker region. Galectin-12 is predominantly expressed in adipose tissue, but is also detected in macrophages and other leukocytes. Downregulation of galectin-12 in mouse 3T3-L1 cells impairs their differentiation into adipocytes. Conversely, overexpression of galectin-12 in vitro induces cell cycle arrest in G1 and apoptosis. Upregulation of galectin-12 and initiation of G1 cell cycle arrest are associated with driving pre-adipocytes toward terminal differentiation. Galectin-12 deficiency increases insulin sensitivity and glucose tolerance in obese animals. Galectin-12 inhibits macrophage polarization to the M2 population, enhancing inflammation and decreasing insulin sensitivity in adipocytes. Galectin-12 also affects myeloid differentiation, which is associated with chemotherapy resistance. In addition to highlighting the above-mentioned aspects, this review also discusses the potential clinical applications of modulating the function of galectin-12.

Ultraviolet irradiation promotes FOXP3 transcription via p53 in psoriasis.

The decrease of forkhead box P3-positive (FOXP3 + ) regulatory T cells (Tregs) causes an immune imbalance with effector T cells in psoriasis. Previous studies have demonstrated that in addition to its known effects on keratinocytes and effector T cells, ultraviolet (UV) irradiation alleviates psoriasis via the upregulation of FOXP3 + Tregs. However, the mechanism is unclear. Here, we found that FOXP3 + T cells were increased in psoriatic lesions after UVB irradiation (t' = 3.7006, P < 0.01), as determined by immunohistochemical staining. In addition, the levels of FOXP3 and p53, one of the downstream targets of UV irradiation, showed accordant changes after UV irradiation. Experiments that used a MAPK inhibitor, p53 mutant cell lines, p53 inhibitor and p53 shRNA showed a decrease in FOXP3 levels, suggesting that p53 is required for UV-induced FOXP3 transcription. Next, we demonstrated that there are two binding sites for p53 on FOXP3 by informatics tools, a dual-luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay. One binding site (-1771 to -1583) is located at the promoter region and is adjacent to a previously reported p53-binding region in breast cancer cells. The other (+3845 to +4042) is located within the first intron and has not been previously reported. Our study demonstrated that FOXP3 is regulated, at least in part, by the binding of p53 to several binding sites in the promoter and intron regions following UV irradiation in psoriasis. It will be helpful to further clarify the regulatory mechanism of FOXP3 transcription and to provide new insights into the mechanisms that mediate the effects of UV irradiation in autoimmune skin disorders.

Ablation of a galectin preferentially expressed in adipocytes increases lipolysis, reduces adiposity, and improves insulin sensitivity in mice.

The breakdown of triglycerides, or lipolysis, is a tightly controlled process that regulates fat mobilization in accord with an animal's energy needs. It is well established that lipolysis is stimulated by hormones that signal energy demand and is suppressed by the antilipolytic hormone insulin. However, much still remains to be learned about regulation of lipolysis by intracellular signaling pathways in adipocytes. Here we show that galectin-12, a member of a ß-galactoside-binding lectin family preferentially expressed by adipocytes, functions as an intrinsic negative regulator of lipolysis. Galectin-12 is primarily localized on lipid droplets and regulates lipolytic protein kinase A signaling by acting upstream of phosphodiesterase activity to control cAMP levels. Ablation of galectin-12 in mice results in increased adipocyte mitochondrial respiration, reduced adiposity, and ameliorated insulin resistance/glucose intolerance. This study identifies unique properties of this intracellular galectin that is localized to an organelle and performs a critical function in lipid metabolism. These findings add to the significant functions exhibited by intracellular galectins, and have important therapeutic implications for human metabolic disorders.

Galectin-9 blockade synergizes with ATM inhibition to induce potent anti-tumor immunity.

Although current cancer immunotherapies that target PD-1/PD-L1 immune checkpoint to reinvigorate exhausted T cells have achieved impressive clinical outcomes, only a small proportion of patients respond. New therapeutic targets are therefore needed to be identified to further unleash the anti-tumor potential of T cells and benefit more patients. Galectin-9 (Gal-9), initially identified as a ligand for TIM-3 to induce T cell death, acts as an immunosuppressive regulator in the tumor microenvironment (TME) but its potential as a therapeutic target remains largely elusive. Here we show that antibody neutralization of Gal-9, in combination with inhibition of Ataxia telangiectasia mutated (ATM), a kinase essential for DNA damage response (DDR), is a promising modality for cancer immunotherapy. Genetic depletion of ATM in tumors markedly potentiated anti-Gal-9 therapy in a syngeneic mouse model. Mechanistically, ATM inhibition greatly upregulated Gal-9 expression and secretion in a variety of human and murine tumor cells via the cGAS-STING-interferon ß (IFNß) innate immune pathway. Combination of Gal-9 inhibition with AZD1390, a selective ATM inhibitor currently evaluated in clinical trials, significantly suppressed tumor growth and prolonged survival in multiple syngeneic mouse models, including the poorly-immunogenic LLC lung tumors that do not respond to PD-1/PD-L1 blockade, concomitant with increased T cell infiltration. These results reveal Gal-9 induction via STING/IFNß signaling as an important mechanism mediating tumor immune escape that could be targeted for cancer immunotherapies, and unveil a novel anti-Gal-9-based combination strategy for cancer immunotherapies in a wide variety of malignancies, including those resistant to PD-1/PD-L1 blockade.

The Critical Role of Galectin-12 in Modulating Lipid Metabolism in Sebaceous Glands.

Sebaceous glands play an important role in maintaining the skin barrier function by producing lipids. Dysregulated lipid production in these glands may contribute to the pathogenesis of human skin diseases. Galectin-12, a member of the ß-galactoside‒binding lectin family, is preferentially expressed in adipocytes, where it regulates adipogenesis and functions as an intrinsic negative regulator of lipolysis. It is also expressed by sebocytes and contributes to the proliferation of this cell type. In this study, we show the association between galectin-12 expression and sebocyte differentiation. Galectin-12 knockdown in a human sebocyte cell line reduced lipogenesis and decreased the production of cholesteryl esters, triglycerides, free fatty acids, and cholesterol. Metabolomic analysis of skin surface lipids showed that the levels of the lipids mentioned earlier decreased in sebaceous gland‒specific galectin-12‒knockout mice compared with that in wild-type mice. In addition, galectin-12 positively regulated peroxisome proliferator‒activated receptor-γ transcriptional activity in sebocytes stimulated with fatty acids. Downregulating galectin-12 suppressed the expression of peroxisome proliferator‒activated receptor-γ target genes-acetyl-coenzyme A synthetase 2 gene ACS2 and diacylglycerol O-acyltransferase 1 gene DGAT1-that are required for fatty acid activation and cholesterol and triglyceride biosynthesis. In conclusion, galectin-12 is a positive regulator of sebaceous lipid metabolism with a potential role in the maintenance of skin homeostasis.

Fc-competent multispecific PDL-1/TIGIT/LAG-3 antibodies potentiate superior anti-tumor T cell response.

The landscape of current cancer immunotherapy is dominated by antibodies targeting PD-1/PD-L1 and CTLA-4 that have transformed cancer therapy, yet their efficacy is limited by primary and acquired resistance. The blockade of additional immune checkpoints, especially TIGIT and LAG-3, has been extensively explored, but so far only a LAG-3 antibody has been approved for combination with nivolumab to treat unresectable or metastatic melanoma. Here we report the development of a PDL1 × TIGIT bi-specific antibody (bsAb) GB265, a PDL1 × LAG3 bsAb GB266, and a PDL1 × TIGIT × LAG3 tri-specific antibody (tsAb) GB266T, all with intact Fc function. In in vitro cell-based assays, these antibodies promote greater T cell expansion and tumor cell killing than benchmark antibodies and antibody combinations in an Fc-dependent manner, likely by facilitating T cell interactions (bridging) with cancer cells and monocytes, in addition to blocking immune checkpoints. In animal models, GB265 and GB266T antibodies outperformed benchmarks in tumor suppression. This study demonstrates the potential of a new generation of multispecific checkpoint inhibitors to overcome resistance to current monospecific checkpoint antibodies or their combinations for the treatment of human cancers.

Inhibition of Galectin-9 sensitizes tumors to anthracycline treatment via inducing antitumor immunity.

Anthracyclines are a class of conventionally and routinely used first-line chemotherapy drugs for cancer treatment. In addition to the direct cytotoxic effects, increasing evidence indicates that the efficacy of the drugs also depends on immunomodulatory effects with unknown mechanisms. Galectin-9 (Gal-9), a member of the ß-galactoside-binding protein family, has been demonstrated to induce T-cell death and promote immunosuppression in the tumor microenvironment. Here, we asked whether anthracycline-mediated immunomodulatory activity might be related to Gal-9. We found that combining doxorubicin with anti-Gal-9 therapy significantly inhibited tumor growth and prolonged overall survival in immune-competent syngeneic mouse models. Moreover, Gal-9 expression was increased in response to doxorubicin in various human and murine cancer cell lines. Mechanistically, doxorubicin induced tumoral Gal-9 by activating the STING/interferon ß pathway. Clinically, Gal-9 and p-STING levels were elevated in the tumor tissues of breast cancer patients treated with anthracyclines. Our study demonstrates Gal-9 upregulation in response to anthracyclines as a novel mechanism mediating immune escape and suggests targeting Gal-9 in combination with anthracyclines as a promising therapeutic strategy for cancer treatment.

Galectin-3 negatively regulates TCR-mediated CD4+ T-cell activation at the immunological synapse.

We have investigated the function of endogenous galectin-3 in T cells. Galectin-3-deficient (gal3(-/-)) CD4(+) T cells secreted more IFN-gamma and IL-4 than gal3(+/+)CD4(+) T cells after T-cell receptor (TCR) engagement. Galectin-3 was recruited to the cytoplasmic side of the immunological synapse (IS) in activated T cells. In T cells stimulated on supported lipid bilayers, galectin-3 was primarily located at the peripheral supramolecular activation cluster (pSMAC). Gal3(+/+) T cells formed central SMAC on lipid bilayers less effectively and adhered to antigen-presenting cells less firmly than gal3(-/-) T cells, suggesting that galectin-3 destabilizes the IS. Galectin-3 expression was associated with lower levels of early signaling events and phosphotyrosine signals at the pSMAC. Additional data suggest that galectin-3 potentiates down-regulation of TCR in T cells. By yeast two-hybrid screening, we identified as a galectin-3-binding partner, Alix, which is known to be involved in protein transport and regulation of cell surface expression of certain receptors. Co-immunoprecipitation confirmed galectin-3-Alix association and immunofluorescence analysis demonstrated the translocation of Alix to the IS in activated T cells. We conclude that galectin-3 is an inhibitory regulator of T-cell activation and functions intracellularly by promoting TCR down-regulation, possibly through modulating Alix's function at the IS.

Phosphorylation and Stabilization of PD-L1 by CK2 Suppresses Dendritic Cell Function.

Targeting immune checkpoints such as programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) has transformed cancer treatment, with durable clinical responses across a wide range of tumor types. However, a high percentage of patients fail to respond to anti-PD-1/PD-L1 treatment. A greater understanding of PD-L1 regulation is critical to improving the clinical response rate of PD-1/PD-L1 blockade. Here, we demonstrate that PD-L1 is phosphorylated and stabilized by casein kinase 2 (CK2) in cancer and dendritic cells (DC). Phosphorylation of PD-L1 at Thr285 and Thr290 by CK2 disrupted PD-L1 binding with speckle-type POZ protein, an adaptor protein of the cullin 3 (CUL3) ubiquitin E3 ligase complex, protecting PD-L1 from CUL3-mediated proteasomal degradation. Inhibition of CK2 decreased PD-L1 protein levels by promoting its degradation and resulted in the release of CD80 from DC to reactivate T-cell function. In a syngeneic mouse model, combined treatment with a CK2 inhibitor and an antibody against T-cell immunoglobulin mucin-3 (Tim-3) suppressed tumor growth and prolonged survival. These findings uncover a mechanism by which PD-L1 is regulated and suggest a potential antitumor treatment option to activate DC function by blocking the CK2-PD-L1 pathway and inhibiting Tim-3. SIGNIFICANCE: This work identifies a role for CK2 in immunosuppression by phosphorylation and stabilization of PD-L1, identifying CK2 inhibition as an immunotherapeutic approach for treating cancer.

Activated T cell-derived exosomal PD-1 attenuates PD-L1-induced immune dysfunction in triple-negative breast cancer.

Programmed cell death 1 (PD-1) is widely expressed in tumor-infiltrating lymphocytes (TILs) of triple-negative breast cancer (TNBC). As a dominant inhibitory immune checkpoint (ICP) receptor, cell surface PD-1 is well-known to transduce negative signaling of effector T cell activity during cell-cell contact. However, despite its well-documented inhibitory effects, higher PD-1 expression in TILs is significantly associated with longer survival in TNBC patients. This phenomenon raises an interesting question whether PD-1 harbors positive activity to enhance anti-tumor immunity. Here, we show that PD-1 is secreted in an exosomal form by activated T cells and can remotely interact with either cell surface or exosomal programmed death-ligand 1 (PD-L1), induce PD-L1 internalization via clathrin-mediated endocytosis, and thereby prevent subsequent cellular PD-L1: PD-1 interaction, restoring tumor surveillance through attenuating PD-L1-induced suppression of tumor-specific cytotoxic T cell activity. Our results, through revealing an anti-PD-L1 function of exosomal PD-1, provide a positive role to enhance cytotoxic T cell activity and a potential therapeutic strategy of modifying the exosome surface with membrane-bound inhibitory ICP receptors to attenuate the suppressive tumor immune microenvironment.

Galectin-9 interacts with PD-1 and TIM-3 to regulate T cell death and is a target for cancer immunotherapy.

The two T cell inhibitory receptors PD-1 and TIM-3 are co-expressed during exhausted T cell differentiation, and recent evidence suggests that their crosstalk regulates T cell exhaustion and immunotherapy efficacy; however, the molecular mechanism is unclear. Here we show that PD-1 contributes to the persistence of PD-1+TIM-3+ T cells by binding to the TIM-3 ligand galectin-9 (Gal-9) and attenuates Gal-9/TIM-3-induced cell death. Anti-Gal-9 therapy selectively expands intratumoral TIM-3+ cytotoxic CD8 T cells and immunosuppressive regulatory T cells (Treg cells). The combination of anti-Gal-9 and an agonistic antibody to the co-stimulatory receptor GITR (glucocorticoid-induced tumor necrosis factor receptor-related protein) that depletes Treg cells induces synergistic antitumor activity. Gal-9 expression and secretion are promoted by interferon ß and γ, and high Gal-9 expression correlates with poor prognosis in multiple human cancers. Our work uncovers a function for PD-1 in exhausted T cell survival and suggests Gal-9 as a promising target for immunotherapy.

The stabilization of PD-L1 by the endoplasmic reticulum stress protein GRP78 in triple-negative breast cancer.

The immune checkpoint blockade therapy has emerged as encouraging treatment strategies in various cancer types. Anti-PD-L1 (programmed death-ligand 1) antibodies have been approved for triple-negative breast cancer, however the response rate yet to be optimized. It would be imperative to further understand and investigate the molecular mechanisms of PD-L1 regulation. Here, we identified glucose regulatory protein 78 (GRP78), a major endoplasmic reticulum (ER) stress responding protein, as a novel binding partner of PD-L1. GRP78 interacts with PD-L1 at the ER region and increases PD-L1 levels via regulating its stability. ER stress, triggered by different stimuli such as conventional chemotherapy, leads to the induction of PD-L1 in a GRP78-dependent manner. We showed that GRP78 modulates the response to chemotherapy, and dual-high levels of GRP78 and PD-L1 correlates with poor relapse-free survival in triple-negative breast cancer. Altogether, our study provides novel molecular insights into the regulatory mechanism of PD-L1 by revealing its interaction with GRP78, and offers a rationale to target GRP78 as a potential therapeutic strategy to enhance anti-tumor immunity.

Erratum: The stabilization of PD-L1 by the endoplasmic reticulum stress protein GRP78 in triple-negative breast cancer.

[This corrects the article on p. 2621 in vol. 10, PMID: 32905506.].

Targeting Glycosylated PD-1 Induces Potent Antitumor Immunity.

Immunotherapies targeting programmed cell death protein 1 (PD-1) and programmed cell death 1 ligand 1 (PD-L1) immune checkpoints represent a major breakthrough in cancer treatment. PD-1 is an inhibitory receptor expressed on the surface of activated T cells that dampens T-cell receptor (TCR)/CD28 signaling by engaging with its ligand PD-L1 expressed on cancer cells. Despite the clinical success of PD-1 blockade using mAbs, most patients do not respond to the treatment, and the underlying regulatory mechanisms of PD-1 remain incompletely defined. Here we show that PD-1 is extensively N-glycosylated in T cells and the intensities of its specific glycoforms are altered upon TCR activation. Glycosylation was critical for maintaining PD-1 protein stability and cell surface localization. Glycosylation of PD-1, especially at the N58 site, was essential for mediating its interaction with PD-L1. The mAb STM418 specifically targeted glycosylated PD-1, exhibiting higher binding affinity to PD-1 than FDA-approved PD-1 antibodies, potently inhibiting PD-L1/PD-1 binding, and enhancing antitumor immunity. Together, these findings provide novel insights into the functional significance of PD-1 glycosylation and offer a rationale for targeting glycosylated PD-1 as a potential strategy for immunotherapy. SIGNIFICANCE: These findings demonstrate that glycosylation of PD-1 is functionally significant and targeting glycosylated PD-1 may serve as a means to improve immunotherapy response.

Galectins: structure, function and therapeutic potential.

Galectins are a family of animal lectins that bind beta-galactosides. Outside the cell, galectins bind to cell-surface and extracellular matrix glycans and thereby affect a variety of cellular processes. However, galectins are also detectable in the cytosol and nucleus, and may influence cellular functions such as intracellular signalling pathways through protein-protein interactions with other cytoplasmic and nuclear proteins. Current research indicates that galectins play important roles in diverse physiological and pathological processes, including immune and inflammatory responses, tumour development and progression, neural degeneration, atherosclerosis, diabetes, and wound repair. Some of these have been discovered or confirmed by using genetically engineered mice deficient in a particular galectin. Thus, galectins may be a therapeutic target or employed as therapeutic agents for inflammatory diseases, cancers and several other diseases.

Inhibition of ATR downregulates PD-L1 and sensitizes tumor cells to T cell-mediated killing.

The ataxia telangiectasia and Rad3-related (ATR) kinase plays a crucial role in maintaining genome stability in response to DNA damage. Once activated, ATR acts via its downstream target to arrest the cell cycle, promote DNA repair, and enhance cell survival. Therefore, ATR has become an attractive therapeutic target in cancer therapy. Multiple clinical studies have demonstrated that ATR inhibitors can sensitize cancer cells to conventional DNA damaging agents. However, the potential effects of ATR inhibitors on immune response in the tumor microenvironment, especially on the expression of immune checkpoint-related proteins, remain elusive. Here we show that DNA damaging agents, such as ionizing radiation and cisplatin, significantly induce cell surface PD-L1 expression in various cancer cell types. This effect is blocked by depletion or pharmacological inhibition of ATR, suggesting the essential role of ATR in DNA damage-induced PD-L1 expression. Mechanistically, we show that disruption of ATR destabilizes PD-L1 in a proteasome-dependent manner. Furthermore, clinical ATR kinase inhibitor downregulates PD-L1 expression to attenuate PD-L1/PD-1 interaction and sensitize cancer cells to T cell killing. Collectively, our findings indicate that in addition to potentiating DNA damage, ATR inhibitor concurrently downregulates PD-L1 levels and enhances anti-tumor immune responses. Moreover, our data reveal a potential crosstalk between DNA damage response signaling and immune checkpoints, providing a rationale for the combination therapy of ATR inhibitor and immune checkpoint blockade.

The role of T-cell immunoglobulin mucin-3 and its ligand galectin-9 in antitumor immunity and cancer immunotherapy.

Cancer treatment in the past few years has been transformed by a new kind of therapy that targets the immune system instead of the cancer itself to reinvigorate antitumor immunity with astonishing results. However, primary and acquired resistance to this type of treatment, namely immune checkpoint blockade (ICB), continue to counter treatment efficacy. In many cases, resistance has been attributed to defective or chronically enhanced interferon signaling and/or upregulation of alternative immune checkpoints, including T-cell immunoglobulin mucin-3 (Tim-3) and its ligand galactin-9 (Gal-9). In this article, we briefly describe the current knowledge of common checkpoint resistance mechanisms, focusing on the Tim-3/Gal-9 pathway as an alternative checkpoint that holds great promise as another target for ICB.